Hops, in the form of either the ground dried plant or an extract, are used in brewing to give malt beverages, such as beer or ale, their characteristic bitter flavor and pleasant aroma. The hops or a hop extract may be added to boiling wort in the brew kettle. An isomerized hop extract, if it is highly purified, may be added post kettle, i.e., after the wort has been boiled or after fermentation. The primary hop constituents used in the brewing process are the alpha acids, the beta acids, the uncharacterized soft resins and the hop oils. The alpha acids are known as humulones and the beta acids are known as lupulones. The alpha acids are the precursors of the bitter substances in beer. The beta acids or lupulones have low solubility in kettle wort and beer and play a relatively minor role in the brewing process.
During brewing, chemical changes are made in the humuones resulting in the formulation of compounds known as iso-alpha acids, i.e., isohumulone, isocohumulone and isoadhumulone. These iso-alpha acids are formed in the kettle during the kettle boil in the normal brewing process and are the primary contributors to the characteristic bitter flavor of beer and ale.
Hop extracts have been used in brewing beer for a number of years. The reasons are several fold. When whole hops are added to the kettle, the yield of isohumulone is poor, e.g., 20-25% based on the humulone present in the hops. However, the conversion of humulones in a hop extract to isohumulones can be very high, e.g. 80%. Furthermore, the utilization of the pure isohumulones in a pre-isomerized extract which is added post kettle is known to be extremely high, e.g. 70-90%.
In order to use a hop extract post kettle, it must contain isohumulones of a high degree of purity. It should not contain hop components such as lupulones, waxes and other insoluble residues which can cause substantial haze, i.e., turbidity or gushing, i.e., rapid carbon dioxide release.
Extracts containing isohumulones of only 80% purity, for example, cannot be added post kettle in amounts exceeding approximately 10-15 ppm of isohumulone without the possibility of causing turbidity in the finished product. On the other hand, extracts containing isohumulone of high purity, .gtoreq.90% for example, can be added post kettle at levels exceeding 20 ppm without a significant increase in turbidity.
It is known that isohumulone derived from hops or an unreduced hop extract can cause light instability in malt beverages. The exposure of such a beer or ale to light can result in the beverage becoming "light struck" and having a skunky odor.
For many years researchers have been striving to produce an anactinic or light stable hop extract.
In Koch et al., U.S. Pat. No. 3,044,879, a method for the isomerization and reduction of alpha acids to produce reduced isohumulones is disclosed and in the Westermann et al., U.S. Pat. No. 3,558,326, a method is disclosed for the production of a post-kettle additive and a kettle additive.
The hopping materials produced by the methods of the Koch et al. and Westermann et al. patents gave a product which produced beers that were far better with respect to light stability than beers produced with unreduced isohumulone (conventional beers). However, a significant residual light instability remained which was attributed to residual unreduced isohumulone.
Subsequently, with the development of high performance liquid chromatography (HPLC) techniques for the analysis of unreduced isohumulone in reduced hop extracts, it was possible to tell that products produced by the dual-phase isomerization/reduction methods of Koch et al. and Westermann et al. contained anywhere from 2-5% unreduced isohumulone (based on total isohumulones present).
In the Goldstein et al. U.S. Pat. No. 4,324,810, a method is disclosed for making an anactinic hop kettle additive and a purified anactinic post-kettle additive without the use of organic solvents. This single phase isomerization and reduction process was carried out at 60.degree.-65.degree. C. (similar to the prior art); the product still contained 1-3% unreduced isohumulone based on the total isohumulones present. The starting material for the process reported in Goldstein et al. was a liquid carbon dioxide hop extract containing alpha acids, beta acids, hop oils, and waxes.
Research subsequent to the Goldstein et al. patent found that the amount of unreduced isohumulone could be brought below the detection limit of the HPLC technique (0.5% unreduced isohumulones based on the total isohumulones) by carrying out the isomerization/reduction at higher temperatures (80.degree.-100.degree. C.). Although beers produced with these hop extracts containing less than 0.5% unreduced isohumulones were more light stable than those made with extracts produced by the dual phase methods (containing from 2-5% unreduced isohumulones) or by the low temperature single phase method, analyses by both sensory and chromatographic techniques surprisingly indicated that a residual light instability was still present.
It was theorized that the residual light instability in the single phase products might be due to one of the following:
1. The analytical techniques for the estimation of unreduced isohumulone in the hop extracts were not giving reliable readings and there was, in fact, more than 0.5% unreduced isohumulone present in these extracts.
2. Compounds other than the unreduced isohumulones were precursors of 3-methyl-2-butene-1-thiol (which is the malodorous end-product of the light-induced reaction and is the immediate cause of "light struck" aroma) and were the underlying cause of the light instability.
3. High levels of reduced isohumulone caused light instability.
4. Reduced isohumulone was oxidized to isohumulone before or after addition to beer. This re-formed isohumulone caused the light instability.
Subsequent research showed that the cause of light instability in beer bittered with extracts produced by the high temperature single phase method was due to compounds other than unreduced isohumulones (theory 2). Briefly, it was shown that there was very little or no residual 3-methyl-2-butene-1-thiol, the chemical responsible for the "skunky" odor, detected (either by sensory or analytical methods) when pure crystalline unreduced trans-isohumulone was added to beer at levels equivalent to or less than 0.5% (based on total isohumulones). In addition, attempts to oxidize reduced isohumulone under brewing conditions met with failure. Furthermore, although it was shown experimentally that beers bittered with reduced isohumulone could yield 3-methyl-2-butene-1-thiol under conditions of high intensity ultraviolet irradiation in quartz vessels, this reaction did not occur when using visible light in flint glass containers.
We believe that there may be some non-isohumulone light unstable products (NILUPS) in the hop extracts which are the cause of the residual light instability.
It has been well established in the literature that the enclosed side chain containing carbonyl number 1 and a non-conjugated double bond (See Diagram) must be intact for 3-methyl-2-butene-1-thiol formation (under conditions of visible light in a flint glass container) and the condition (degree of reduction-oxidation) of carbonyls numbers 2, 3, or enol 4 would not directly affect light stability The NILUPS described above still contain the unreduced carbonyl 1 and the non-conjugated double bond; compounds containing this moiety are the cause of the light instability found in products brewed with hop extracts made by the single-phase high temperature isomerization/reduction of isohumulone.
We do not know with certainty which light unstable compounds are present or how they might be formed. However, as can be seen in the diagram, isohumulones can exist in two geometric forms, cis and trans. The aqueous alkaline reduction of carbonyl 1 in trans-isohumulone proceeds at a much lower rate than the same reduction of carbonyl 1 in cis-isohumulone. The lower reduction rate of trans-isohumulone explains the incomplete reduction of carbonyl 1, which could be responsible for products that are light sensitive. ##STR1##
Another possible cause of inhibition of reduction of the carbonyl 1 is a side reaction involving the other carbonyls. If any or all of these carbonyl groups are reduced to secondary alcohols, for example, the pK.sub.a (s) of the resulting compound(s) would be higher than the reactant (isohumulone) or for that matter reduced isohumulone. This decrease in acidity would lower the solubility of the resulting compound(s) in alkaline sodium borohydride (depending on the pH of that solution) due to a shift in the free acid- "salt" equilibrium. Lower solubility would cause a further decrease in the reduction rate of carbonyl 1. It is well documented in the literature that the reduction of one of the carbonyl groups (2-4) would also cause a red shift in the UV absorption maximum from approximately 250 nm to approximately 280 nm (in alkaline methyl alcohol). Small amounts of these compounds can be detected by HPLC with UV detection at 280 nm but not at 254 nm, the wavelength commonly used for this procedure.
The solubility of these compounds (NILUPS) in beer would be similar to that of reduced isohumulone. Therefore, their effect on light stability would be comparable to that of unreduced isohumulone since the side chain containing carbonyl 1, the enclosed side chain, is still intact.
In an attempt to push the isomerization/reduction reaction to completion (high temperature reaction) i.e. produce a product with only reduced isohumulone and no unreduced isohumulone, we have instead surprisingly produced a hop extract that contains no unreduced isohumulone (below the HPLC detection limit), but a small amount of NILUPS. Unlike unreduced isohumulone, NILUPS have a different pK.sub.a than reduced isohumulone; separation of the NILUPS from the desired product by careful adjustment of pH is therefore possible.